The time course of molecular acclimation to seawater in a euryhaline fish

Abstract

The Arabian pupfish, Aphanius dispar, is a euryhaline fish inhabiting both inland nearly-freshwater desert ponds and highly saline Red Sea coastal lagoons of the Arabian Peninsula. Desert ponds and coastal lagoons, located respectively upstream and at the mouths of dry riverbeds ("wadies"), have been found to potentially become connected during periods of intense rainfall, which could allow the fish to migrate between these different habitats. Flash floods would therefore flush Arabian pupfish out to sea, requiring a rapid acclimation to a greater than 40 ppt change in salinity. To investigate the molecular pathways of salinity acclimation during such events, a Red Sea coastal lagoon and a desert pond population were sampled, with the latter exposed to a rapid increase in water salinity. Changes in branchial gene expression were investigated via genome-wide transcriptome measurements over time from 6 h to 21 days. The two natural populations displayed basal differences in genes related to ion transport, osmoregulation and immune system functions. These mechanisms were also differentially regulated in seawater transferred fish, revealing their crucial role in long-term adaptation. Other processes were only transiently activated shortly after the salinity exposure, including cellular stress response mechanisms, such as molecular chaperone synthesis and apoptosis. Tissue remodelling processes were also identified as transient, but took place later in the timeline, suggesting their importance to long-term acclimation as they likely equip the fish with lasting adaptations to their new environment. The alterations in branchial functional pathways displayed by Arabian pupfish in response to salinity increases are diverse. These reveal a large toolkit of molecular processes important for adaptation to hyperosmolarity that allow for successful colonization to a wide variety of different habitats.

Figure 1

Sampling locations and target organism. (a) Map of the region with inset (b) showing the specific sampling locations in the central western region of Saudi Arabia. (c) Photos of the sampling sites. (d) Pictures of collected male (top) and female (bottom) Arabian pupfish.

Figure 2

Experimental design. (a) Fish from desert pond (DP) kept in native water salinity (1.9 ppt) were sampled at time 0. (b) Fish from Red Sea lagoon (RS) kept in native water salinity (43 ppt) were sampled at time 0. (c) Fish from desert pond transferred to seawater (43 ppt) were sampled at 5 different time points. A total of 33 samples were analyzed.

Figure 3

Principal component analysis (PCA) of variance stabilized expression values of the 500 most variable genes for the gills of desert pond (n = 4) and Red Sea coastal lagoon (n = 6) Aphanius dispar individuals at time 0. 44% of the total variation is explained by the first two components.

Figure 4

Numbers of differentially expressed genes in pairwise comparisons of desert pond fish controls (0 h) versus seawater exposed fish at different time points (6 h, 24 h, 72 h, 7 d, 21 d) and Red Sea (RS) population, and consecutive post-transfer time points.

Figure 5

Heatmap of transiently (a) or monotonously (b) up-(↑) and down- (↓) regulated differentially expressed genes (FDR corrected p-value < 0.05) over the experimental timeline, as identified by ImpulseDE2 analysis. DP and RS stand for desert pond and Red Sea samples, respectively.

Figure 6

Changes in ratio and expression direction of differentially expressed genes grouped by functions along the acclimation timeline. Circle sizes are proportional to the gene number ratio for a specific function at a certain time point. Circle colours correspond to the ratio of the upregulated (red circles) vs downregulated (blue circles) genes for the function at that time point; the fuller the colour, the higher the proportion of up- or downregulated genes (e.g. 1.0 = all genes are upregulated; 0 = half of the genes are upregulated, half are downregulated; − 1.0 = all genes are downregulated).